1. Field of the Invention
The present invention relates to new diagnostic procedures or to protocols suitable for use in screening candidate therapeutic drug agents. The present invention provides human, living, dynamic experimental models, and metabolic markers related thereto, for examining some aspects of the pathophysiology of familial or non-familial neuro-degenerative diseases selected from the group hereby limited solely to Charcot-Marie-Tooth disease, familial Alzheimer's disease, familial Parkinson's disease, Huntington's disease, spinal muscular atrophy, Friedreich'a ataxia, giant axon neuropathy, juvenile ceroid-lipofuscinosis, familial motor neuron diseases, juvenile diabetic polyneuropathy and Down's syndrome, including various individual genetic subvarieties thereof.
2. Description of Prior Art
J. T. Coyle and P. Puttfarcken (1993), Science 262:689-695 (1993) noted that                There is an increasing amount of experimental evidence that oxidative stress is a causal, or at least an ancillary, factor in the neuropathology of several adult neurodegenerative disorders, as well as in stroke, trauma, and seizures . . . [pg. 689]The authors proceeded to review the various sources and origins of neuronal oxidative stress and reviewed the known intrinsic metabolic mechanisms for natural protection against such stress. They mentioned the ability of vitamin E to inhibit lipid peroxidation (pg. 690, first column, lines 5-9), and they referred to the neuroprotective effect of other antioxidants (i.e., page 692, column one). Notably, on page 690, second column, lines 1-5 the authors also mentioned that        Furthermore, peroxy radicals can combine with an abstracted hydrogen atom to form lipid hydroperoxides which, in the presence of Fe2+, decompose to alkoxy radicals and aldehydes.On page 692 (column three, lines 7-11) the authors specifically mentioned that they were referring to diseases such as Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD). In another statement of particular relevance (page 693, column three, lines 32-39), the authors commented that        . . . notably, protein carbonyl content, a measure of protein oxidation, was elevated by 85% in patents with sporadic ALS as compared to controls, suggesting that oxidative stress is a common feature of ALS whether the disease is due to loss of CuZnSOD activity or to other causes . . .The authors concluded (page 694, column two, lines 5-11), in part        . . . Nevertheless, the evidence, while still largely circumstantial, is convincing that oxidative stress represents an important pathway, initiated in part by Glu, that leads to neuronal degeneration in a manner consistent with the course and pathology of several degenerative disorders of the brain . . .        
One aspect of cellular oxidative stress is the pathological formation of crosslinked proteins. As the H polypeptide chains of neurofilaments have an especially high lysine content (20%), neurofilaments are particularly susceptable to spurious crosslinking reactions which may be induced by lipid peroxidation products [Carden et al., Neurochem. Pathol. 5:25-35 (1986)]. The results of several published research studies suggest that dysfunctional lipid peroxidation may be a contributing factor in the etiology of Parkinson's disease [Fahn, Ann. NY Acad. Sci. 570:186-196 (1989)], multiple sclerosis [Hunter et al., Neurochem. Res. 10:1645-1652 (1985)] and Duchenne muscular dystrophy [Kar and Pearson, Clin. Chim. Acta 94:277-280 (1979); Jackson et al., Med. Biol. 62:135-138 (1984); Hunter and Mohamed, Clin. Chim. Acta 155:123-132 (1986)].
A considerable body of prior art has provided evidence suggesting that the etiologies of certain neurodegenerative diseases include evidence of chemical crosslinking of neurofilaments. Such studies include work on Charcot-Marie-Tooth (CMT) genetic neuropathies [Hughes and Brownell, J. Neurol. Neurosurg. Psych. 35:648-657 (1972); Brimijoin et al., Science 180:1295-1297 (1973); van Weerden et al., Muscle & Nerve 5:185-196 1982; and Goebel et al., Ital. J. Neurol. Sci. 7:325-332 1986)], giant axon neuropathy [Prineas et al., J. Neuropathol. Exp. Neurol. 35:458-470 (1976)], diabetic polyneuropathy [Yamamura et al., in Diabetic Neuropathy, Goto, Y, sr, ed. (Princeton, Excerpta Medica, (1982) pp. 80-85; Sidenius and Jakobsen, Diabetes 31:689-693 (1982); and Tomlinson and Mayer, J. Auton. Pharmac. 4:59-72 (1984)], Alzheimer's disease [Wisniewski et al., J. Neuropath. Exp. Neurol. 29:163-176 (1970); and Wisniewski et al, in Aging and Cell Structure, volume 1, Johnson, Jr., J E, ed. (New York, Plenum Press, 1982) pp. 110-112], Down's syndrome [Goodison et al., Soc. Neurosci. Abstr. 15(pt. 2): 329 (abstract 135.6) (1989)], Pick's disease [Yoshimura, Clin. Neuropath. 8:1-6 (1989)], Parkinson's disease [Oppenheimer, in Greenfield's Neuropathology, Blackwood, W and Corsellis, JAN, eds. (Chicago, Year Book Medical Publishers, 1976) pp. 612-614; and Cohan, in Clinical Aspects of Aging, third edition, Reichel, W, ed. (Baltimore, Williams & Wilkens, 1989) pp. 167)], amyotrophic lateral sclerosis [Carpenter, Neurology 18:841-851 (1968)], infantile spinal muscular atrophy [Lee et al., Neuropediatrics 20:107-111 (1989)], Friedreich's ataxia [Lamarche et al., Can. J. Neurol. Sci. 9:137-139 (1982)], and alcoholic polyneuropathy [Appenzeller and Richardson, Neurology (Minneap) 16:1205-1209 (1966)].
Considerable biomedical literature indicates that certain sites on normal proteins and lipids are specific targets for spurious chemical crosslinking, most notably the ε-amino groups of lysine residues in proteins and the amine groups of phosphatidyl-ethanolamine molecules in cell lipid membrane bilayers. These primary amine groups are especially prone to attack by small molecular weight carbonyl-containing hydrocarbons. Such carbonyl-containing molecules may originate by many pathological mechanisms still only partly defined, but, in general, they originate from peroxidation of fatty acids or as by-products of sugar metabolism. A mono-carbonyl specie can bind to a protein or amino-lipid, alter its three dimensional structure and possibly affect its chemical activity. A dicarbonyl hydrocarbon can react with two amine groups, thus making a covalent chemical crosslink. The specific primary pathological changes that underlie this type of deterioration remain largely undefined, but their structural products have been characterized in many respects.
For example, the senile plaques and neurofibrillary tangles typical of Alzheimer's disease consist largely of networks of intermediate size protein filaments helically wound in pairs having a periodicity of 80 nm [Selkoe et al. Science 215:1243-1245 (1982)]. Isolated paired helical filament (PHF) has proven to have remarkable properties of chemical stability. PHF chemical crosslinking bonds are not broken by sodium dodecyl sulfate, β-mercaptoethanol, 9.5 M urea, two percent Triton X-100, one percent NP-40, 6 M guanidine hydrochloride, 0.2 N HCl or 0.2 N NaOH. As heating of PHF in the presence of either reducing agents such as β-mercaptoethanol or detergents such as Triton X-100 or NP-40 did not solubilize PHF, bonds other than disulfide are implicated in amino acid crosslinking of this type of rigid intracellular polymer. This unusual chemical stability has seriously impeded PHF analysis by gel electrophoresis [Selkoe et al. (1982)]. As a postulated mechanism for such unusual crosslinking Selkoe et al. noted that “different protein polymers in senile cataracts, terminally differentiated epidermal cells, and red blood cells are covalently cross-linked by γ-glutamyl-ε-lysine sidechain bridges.” Like PHF, these other protein complexes are insoluble in sodium dodecyl sulfate and not solubilized by reducing agents. Selkoe et al. speculated that such γ-glutamyl-ε-lysine crosslinks may also form pathologically in nerve cells, as human brain contains a transglutaminase capable of acting on normal neurofilament to form an insoluble high molecular weight filamentous polymer.
Kikugawa and Beppu [Chem. Phys. Lipids 44:277-296 (1987)] noted that lipid radicals, hydroperoxides and their secondary products (including various aldehydes and ketones) react with neighboring protein molecules, damaging protein structure and function. Such damage includes formation of fluorescent chromophores, lipid-protein adducts, and protein-protein crosslinks. Using sodium dodecyl sulfate-polyacryl-amide gel electrophoresis, these investigators demonstrated that malonaldehyde (also known as malondialdehyde), a bifunctional molecule having two aldehyde groups, can covalently crosslink proteins. This reaction primarily involves Schiff base formation with protein ε-amino groups on the sidechains of lysine residues. Kikugawa and Beppu (1987) also reported that monofunctional aldehydes such as acetaldehyde, 1-hexanal, 1-heptanal and 2,4-decadienal can also crosslink proteins, generating fluorescent products. This biochemical curiosity still not well understood. Some form of self-condensation may be involved.
A report by Piersanti et al. [Neurobiol. Aging 13:S111 (abstract 437) (1992)] documented an increased susceptibility of Alzheimer's disease patient skin fibroblasts to free radical damage. The Piersanti report, taken together with CMT research findings discussed below, lends credence to the concept that skin fibroblast samples from patients having other neurodegenerative disorders will also show evidence of oxidative stress.
Evidence of increased deposition of lipofuscin in various neurodegenerative diseases has been presented. This observation has been documented in studies on amyotrophic lateral sclerosis [Carpenter (1968)], Guam Parkinsonism-dementia [Tan et al., Clin. Exp. Neurol. 17:227-234 (1981)], Alzheimer's disease [Tsuchida et al., Chem. Phys. Lipids 44:297-325 (1987); Moran and Gomez-Ramos, Soc. Neurosci. Abstr. 15(pt. 2):1039 (abstract 414.8) (1989)], Huntington's disease [Tellez-Nagel et al., J. Neuropathol. Exp. Neurol. 33:308-332 (1974)], Meniere's disease [Ylikoski et al. Arch. Otolaryngol. 106:477-483 (1980)], and juvenile ceroid-lipofuscinosis [Schwendemann, in Ceroid-Lipofuscinosis (Batten Disease), Armstrong, D, sr. ed. (New York, Elsevier Biomedical Press, 1982) pp. 117-136]. Heart lipofuscin has been shown to have the following general composition: lipids, 20-50%; protein, 30-60%; and strongly pigmented resin-like hydrolysis-resistant material, 9-20%. Although the exact nature of the hydrolysis-resistant chemical bonds remains to be unequivically defined, the similarity between lipofuscin fluorescence and that of Schiff bases formed between malonaldehyde and primary amines suggests that similar chemical crosslinks may be part of lipofuscin structure [Tsuchida et al. (1987)].
Another fundamental physiological aspect of metabolic oxidative stress is the induction of genes that code for stress proteins, originally known as heat shock proteins (hsp's). Highly conserved genes for these proteins are present in bacteria, plants, yeast and higher animals [Welch, Sci. Am. 268(5):56-64 (1993, pgs. 61-62)]. A wide variety of environmental stimuli are known to induce the expression of these genes, including brief exposures to elevated temperatures, exposure to toxic metals, alcohols, various metabolic poisons, protein denaturants and conditions which induce ischemia/reperfusion trauma (i.e., oxidant injury). Welch [Sci. Am. 268(5):56-64 (1993, pgs. 61-62)] noted that                In animal studies, researchers have observed the induction of stress responses in both the heart and brain after brief episodes of ischemia and reperfusion . . .        Cells that produce high levels of stress proteins appear better able to survive the ischemic damage than cells that do not . . .To some limited extent, some hsp's are normally (i.e., constitutively) expressed in plant, microbial and animal cells. They play a role in normal protein post-translational processing and normal metabolic turnover of proteins. Yet even constitutive hsp's are stress inducible. Of the several different classes of hsp's, the hsp70 gene family is the most stress-inducible member.        
Heretofore, some thought has been given to the question of what role hsp's might play in the neurodegenerative diseases addressed herein. However, the question has received little previous attention, and only within a quite limited scope. Autosomal dominant familial forms of Parkinson's disease are now well recognized [Gasser et al., Ann. Neurol. 36(3):387-396 (1994)]. However, a Medline database search from 1976 to April 1995 for references addressing familial Parkinson's disease and stress proteins generated no matches. When a Medline database search from 1976 to April 1995 was expanded to include non-familial cases of Parkinson's disease and stress proteins two references were found [Renkawek et al., Acta Neuropath. 87(5):511-519 (1994) and Namba et al. [Japanese] No to shinkei [Brain & Nerve] 43(1):57-60 (1991)]. However, both of these studies were based on analysis of brain tissue and neither disclosed nor anticipated the present invention.
Likewise, numerous publications have reported genetic forms of Alzheimer's disease [Campion et al. Neurology 45(1):80-85 (1995)]. A Medline database search from 1976 to April 1995 for references addressing familial Alzheimer's disease and stress proteins generated only one match [Guillemette et al., J. Neurochem. 47(3):987-997 (1986)], but actually this paper did not mention that any of its Alzheimer patients had a genetic form of the disease. The report by Guillemette et al. described studies on RNA transcripts obtained from post-mortem Alzheimer's brain biopsy samples. They observed elevated levels of hsp mRNA transcripts in brain samples from Alzheimer patients who had fever immediately prior to death. They also studied human brain mRNA translation (i.e., protein) products. Guillemette et al. reported, in part, that                A positive correlation was found between elevated amounts of the 70-kDa protein and an agonal process accompanied by fever. Two-dimensional analysis of the protein products showed two or more polypeptides for each 70-kDa protein band observed in one-dimensional gels . . . The protein patterns resemble those reported for heat-treated HeLa cells (Slater et al., 1981), suggesting the possibility that heat-shock proteins are expressed in human brain during agonal processes accompanied by fever . . . Despite similarities in the fever profile during the agonal process, control (FIGS. 4b and e) and [Huntington's disease] non-Alzheimer dementia-associated brains (FIGS. 4c and f) exhibited higher yields of 70-kDa peptides than those from Alzheimer-afflicted brains . . .        . . . These results indicate that Alzheimer's disease, in the absence of fever, is not associated with heat-shock response. [pg. 993]        . . . To examine the possibility that the primary pathogenic events that initiate Alzheimer's disease may induce heat-shock expression in the absence of fever, we probed total RNA from the neocortex of an otherwise healthy patient with Alzheimer's disease who died quickly from suicide by hypoxia (K513, FIG. 5, lane 3). There was no detectable expression of the [hsp70] heat-shock transcript in this case . . . [pgs. 994-995].        
Hence, Guillemette et al. restricted their study to analysis of brain samples and did not consider the possibility that stress proteins might be expressed preferentially in cultured non-neuronal tissue. They additionally investigated postmortem brain samples from several Huntington's disease patients, which may explain why this paper is considered to be a genetic study in the Medline database system. Yet for this familial neurodegenerative disorder also, Guillemette et al. attributed the expression of hsp70 to the presence of agonal fever (pg. 993). These investigators did not consider Huntington's disease fibroblasts in their studies. Hence, the present invention was not anticipated by Guillemette et al.
A Medline database search from 1976 to April 1995 was expanded to include references addressing non-familial Alzheimer's disease and stress proteins, and several additional reports were found. Yet none disclosed or anticipated the present invention. A representative sampling of these is presented below. The findings of Guillemette and coworkers regarding hsp expression in Alzheimer's disease postmortem brain samples have been independently confirmed by Morrison-Bogorad et al. [J. Neurochem. 64(1): 235-246 (1995)], who noted that                . . . approximately 40% of the [Alzheimer's disease] patients had a recorded fever of > or =39.2 degrees at or near death . . . Levels of hsp70 mRNAs were increased three- to 33-fold in cerebellum of febrile patients compared with levels in patients whose recorded temperatures were < or =37.5 degrees C. . . . These results indicate that a specific agonal stress, namely fever, can increase the levels of heat shock 70 mRNAs in AD brain . . .        
Renkawek and coworkers (1994) reported immunohistochemical and immunoblotting findings which indicated a “highly induced” expression of hsp27 in Alzheimer brain samples. They also noted that hsp27 expression was also induced, albeit to a lesser extent, in brain samples obtained from patients having other types of dementia, such as Parkinson's dementia, multi-infarct dementia and normal pressure hydrocephalus. Shinohara et al. [J. Neuro. Sci. 119(2):203-208 (1993)] reported that increased levels of two other small heat shock proteins, α-B crystallin and hsp28, were also found in Alzheimer brain samples. Using an antibody specific for α-B crystallin to immunostain nerve samples from patients having several neurodegenerative diseases, Lowe et al. [Neuropath. Appl. Neurobiol. 18(4):341-350 (1992)] studied ballooned neurons having excess phosphorylated neurofilaments. They found that ballooned nerve samples obtained from classical Pick's disease cases, Alzheimer's disease cases and motor neuron disease cases all showed “strong diffuse cytoplasmic immunoreactivity.” They concluded that “ . . . α-B crystallin may be involved in aggregation and remodelling of neurofilaments in disease.”
Using cultured neuronal PC12 cells which had been heat shocked by incubation at 45° C. for 30 minutes, Johnson et al. [Annals NY Acad. Sci. 695:194-197 (1993)] reported evidence of induced expression of hsp72, alterations in the phosphorylation and metabolism of amyloid precursor protein (APP), and formation of a stable complex between hsp72 and tau. Johnson et al. concluded that “these results suggest that heat shock proteins may play either a protective or a promoting role in the formation of A68 and/or the amyloidogenic C-terminal fragment of APP.” Speculating along similar conceptual lines, Hoyer [J. Geriatr. Psych. Neurol. 6(1):3-13 (1993)] stated that in the earliest stages of Alzheimer's disease several stress-related physiological abnormalities, such as glycogen accumulation, might induce the expression of hsp's, and that such events might lead to enhanced generation of amyloid precursor protein. Two other reported studies in this general field, the work of Abe et al. [Neurosci. Letters 125(2):169-171 (1991)] which used cultured lymphoblastoid cells and the work of Morandi et al. [Prog. Clin. Biol. Res. 317:819-827 (1989)] which used cultured rat dorsal root ganglial cells, failed to disclose or anticipate the invention embodied herein.
Approximately 10% of patients having amyotrophic lateral sclerosis (ALS), one of the motor neuron diseases, have a familial form of this disease [Marx, Science 259:1393 (1993)]. Familial cases of ALS are now known to result from a defect in the gene which codes for Cu/Zn-binding superoxide dismutase, an enzyme involved in oxidative free radical metabolism. A Medline database search from 1976 to May 1995 for citations regarding ALS and stress proteins generated three matches [i.e., Migheli et al. Neuropathol. Appl. Neurobiol. 20(3):282-289 (1994)]. Yet all three of these reports were based on studies of anterior hom cell neuronal tissue and none disclosed or anticipated the present invention. When an ALS/stress protein Medline database search from 1976 to May 1995 was expanded to include fibroblasts as a main heading three additional studies were listed [Witt et al., J. Neurol. Sci. 126(2):206-212 (1994); Tandan et al., J. Neurol. Sci. 79(1-2):189-203 (1987); and Beach et al., J. Neurol. Sci. 72(1):49-60 (1986)]. However, the Witt paper focused on calcium homeostasis in ALS fibroblasts. The Tandan et al. study focused on DNA repair abilities of ALS fibroblasts. The Beach et al. study focused on collagenase activity in ALS fibroblasts. None of these additional studies included work on stress proteins. Hence none of them disclosed or anticipated the present invention.
The appearance of high molecular weight ubiquitin-protein conjugates under stress protein inducing conditions is a well documented phenomenon [Raboy et al. Acta Biol. Hung. 42(1-3):3-20 (1991), pg. 8]. Ubiquitin-protein conjugates such as ubiquitinated paired helical filaments [Morishima-Kawashima et al. Neuron 10(6): 1151-1160 (1993)] have been described in studies based on analysis of nerve biopsy tissue. Yet a Medline review of ubiquitin prior art has not revealed a previous search for or identification of disease-associated ubiquitin-protein conjugates in fibroblasts obtained from patients having neurodegenerative diseases.
In U.S. Pat. No. 5,348,945 P. A. Berberian et al. described compositions for and methods of treating certain physiological stress-related states by use of an hsp70 protein as a medicinal agent. The invention embodied in U.S. Pat. No. 5,348,945 is beyond the scope of and irrelevant to the practice of the present specification. The methods and compositions of the present invention were neither disclosed nor anticipated by Berberian et al. Indeed, as defined below, the exogenous introduction of an hsp70 protein into any of the methods and compositions of the present invention would only serve to invalidate the findings, thus rendering useless the practice of the present invention.